Structural Characterization of the Cooperativity of Unfolding of a Heterodimeric Protein using Hydrogen Exchange-Mass Spectrometry |
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Institution: | 1. IFM Biology, Linköping University, SE-581 83 Linköping, Sweden;2. Borås Zoo, SE-501 13 Borås, Sweden;3. Instituto de Neuro-Etologia, Universidad Veracruzana, C.P. 91000 Xalapa, Veracruz, Mexico;1. National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bengaluru 560065, India;2. Indian Institute of Science Education and Research, Pune 411008, India |
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Abstract: | Little is known about how the sequence of structural changes in one chain of a heterodimeric protein is coupled to those in the other chain during protein folding and unfolding reactions, and whether individual secondary structural changes in the two chains occur in one or many coordinated steps. Here, the unfolding mechanism of a small heterodimeric protein, double chain monellin, has been characterized using hydrogen exchange-mass spectrometry. Transient structure opening, which enables HX, was found to be describable by a five state N ? I1 ? I2 ? I3 ? U mechanism. Structural changes occur gradually in the first three steps, and cooperatively in the last step. β strands 2, 4 and 5, as well as the α-helix undergo transient unfolding during all three non-cooperative steps, while β1 and the two loops on both sides of the helix undergo transient unfolding during the first two steps. In the absence of GdnHCl, only β3 in chain A of the protein unfolds during the last cooperative step, while in the presence of 1 M GdnHCl, not only β3, but also β2 in chain B unfolds cooperatively. Hence, the extent of cooperative structural change and size of the cooperative unfolding unit increase when the protein is destabilized by denaturant. The naturally evolved two-chain variant of monellin folds and unfolds in a more cooperative manner than does a single chain variant created artificially, suggesting that increasing folding cooperativity, even at the cost of decreasing stability, may be a driving force in the evolution of proteins. |
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Keywords: | monellin non-cooperative and cooperative kinetics free energy of unfolding entropy DcMN"} {"#name":"keyword" "$":{"id":"k0035"} "$$":[{"#name":"text" "_":"double chain monellin MNEI"} {"#name":"keyword" "$":{"id":"k0045"} "$$":[{"#name":"text" "_":"single chain monellin HX-MS"} {"#name":"keyword" "$":{"id":"k0055"} "$$":[{"#name":"text" "_":"hydrogen exchange coupled to mass spectrometry GdnHCl"} {"#name":"keyword" "$":{"id":"k0065"} "$$":[{"#name":"text" "_":"guanidine hydrochloride BEX"} {"#name":"keyword" "$":{"id":"k0075"} "$$":[{"#name":"text" "_":"back-exchange FWHM"} {"#name":"keyword" "$":{"id":"k0085"} "$$":[{"#name":"text" "_":"full width at half maximum ETD"} {"#name":"keyword" "$":{"id":"k0095"} "$$":[{"#name":"text" "_":"electron transfer dissociation |
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